Robustness to perturbation is a fundamental feature of complex organisms. Mutations are the raw material for evolution, yet robustness to their effects is required for species survival. The mechanisms that produce robustness are poorly understood. Nonlinearities are a ubiquitous feature of development that may link variation in development to phenotypic robustness. Here, we manipulate the gene dosage of a signaling molecule, Fgf8, a critical regulator of vertebrate development. We demonstrate that variation in Fgf8 expression has a nonlinear relationship to phenotypic variation, predicting levels of robustness among genotypes. Differences in robustness are not due to gene expression variance or dysregulation, but emerge from the nonlinearity of the genotype–phenotype curve. In this instance, embedded features of development explain robustness differences. How such features vary in natural populations and relate to genetic variation are key questions for unraveling the origin and evolvability of this feature of organismal development.

Lipases are enzymes necessary for the proper distribution and utilization of lipids in the human body. Lipoprotein lipase (LPL) is active in capillaries, where it plays a crucial role in preventing dyslipidemia by hydrolyzing triglycerides from packaged lipoproteins. Thirty years ago, the existence of a condensed and inactive LPL oligomer was proposed. Although recent work has shed light on the structure of the LPL monomer, the inactive oligomer remained opaque. Here we present a cryo-EM reconstruction of a helical LPL oligomer at 3.8-Å resolution. Helix formation is concentration-dependent, and helices are composed of inactive dihedral LPL dimers. Heparin binding stabilizes LPL helices, and the presence of substrate triggers helix disassembly. Superresolution fluorescent microscopy of endogenous LPL revealed that LPL adopts a filament-like distribution in vesicles. Mutation of one of the helical LPL interaction interfaces causes loss of the filament-like distribution. Taken together, this suggests that LPL is condensed into its inactive helical form for storage in intracellular vesicles.

These findings provide no evidence of either physical or physiological benefits of wearing these suits during submaximal freestyle swimming.

No abstract

This article is available online at http://www.jlr.org (1). Together, these actions make LPL a vital enzyme for the clearance of triglycerides from circulation (2, 3). LPL deficiency (LPLD) is a rare but serious condition caused by homozygous or combined heterozygous loss-of-function mutations in LPL (4). Individuals with LPLD suffer from pronounced accumulation of triglyceride-rich lipoproteins in the plasma, which puts affected individuals at high risk for recurrent, acute pancreatitis (5). Apart from strictly limiting dietary fat, the only treatment option was a gene therapy product known as Glybera, which consists of a gain-of-function LPL variant (LPL S447X) delivered via adeno-associated virus vectors (6). However, at a cost of close to $1 million per patient, Glybera proved too expensive and was withdrawn from the market (7). Volanesorsen, an antisense oligonucleotide drug targeting ApoCIII, decreased triglycerides in individuals with familial chylomicronemia syndrome, but is not currently Food and Drug Administration (FDA)-approved (8). Additional treatment options for LPL-related diseases are therefore needed. Protein therapeutics are a key class of medicines utilized to treat multiple diseases. FDA-approved therapeutics include monoclonal antibodies, replacement enzymes, coagulation factors, hormones, and other plasma proteins. A classic example of this technology is the use of purified insulin for the treatment of both T1D and T2D, which has been in use for nearly 100 years (9). There are also a number of metabolic disorders that have enzyme-replacement therapy (ERT) treatments such as Gaucher's disease, Fabry disease, and Wolman disease (10). Although there are Abstract LPL is a secreted enzyme that hydrolyzes triglycerides from circulating lipoproteins. Individuals lacking LPL suffer from severe hypertriglyceridemia, a risk factor for acute pancreatitis. One potential treatment is to administer recombinant LPL as a protein therapeutic. However, use of LPL as a protein therapeutic is limited because it is an unstable enzyme that is difficult to produce in large quantities. Furthermore, these considerations also limit structural and biochemical studies that are needed for large-scale drug discovery efforts. We demonstrate that the yield of purified LPL can be dramatically enhanced by coexpressing its maturation factor, LMF1, and by introducing novel mutations into the LPL sequence to render it resistant to proteolytic cleavage by furin. One of these mutations introduces a motif for addition of an N-linked glycan to the furin-recognition site. Furin-resistant LPL has previously been reported, but is not commonly used. We show that our modifications do not adversely alter LPL's enzymatic activity, stability, or in vivo function. Together, these data show that furin-resistant LPL is a useful reagent for both biochemical and biomedical studies.